Project Summary Our ability to identify and generate new immunotherapeutics is limited by the challenges associated with recombinant antibody production, particularly correct formation of disulfide bonds which are essential for biological activity. Using a new in vivo crosslinking chemistry to replace native disulfide bonds, the current proposal seeks to overcome this problem and establish a new high throughput platform for stabilization and maturation of monoclonal antibodies. This proposal will make use of translational machinery I have previously developed which allows the highly efficient site specific incorporation of L-DOPA into recombinant proteins. Preliminary data from model proteins confirms formation of covalent cysteinyl-DOPA crosslinks which can be used to replace disulfide bonds. During the K99 period I will define the protein microenvironment required for efficient crosslink formation and also develop new conjugation chemistry to attach cytotoxic small molecules to L-DOPA functionalized antibodies. This will lead to the development of stabilized, disulfide free monoclonal antibodies capable of carrying cytotoxic payloads directly to tumors. Starting in the K99 phase and continuing into the R00 phase, I will combine the stabilized monoclonal antibodies with emulsion-PCR selection methods developed in the mentor's laboratory to establish a rapid high-throughput system for optimizing antibody stability and affinity, as well as performing de novo antibody selections. Ultimately, the efficacy of these improved and functionalized antibodies will be validated in vivo using mouse xenograft models of EGFR+/CD109+ pancreatic and basal-like breast cancer. As a postdoctoral researcher I have established an innovative research program which has already resulted in the development of unique biological tools, high quality research publications and collaborations with research groups at the University of Texas at Austin and Harvard University. Receiving support from the NIH, through the K99/R00 award, will allow me to take advantage of both the extensive resources at the University of Texas at Austin and the experience of three mentors who have strong track records and diverse research interests. This would place me in the best possible position to successfully transition from postdoctoral work to an independent research position. The proposed research will greatly advance the field chemical biology by providing insights into how proteins can be altered to accommodate new amino acid chemistries, while also developing approaches for generating more robust and specific monoclonal antibodies for immunotherapy applications. This proposal will establish a strong multidisciplinary foundation for my own research program developing semi-synthetic peptide and protein therapeutics using exotic and orthogonal amino acid chemistries.